Deficiency of rice hexokinase HXK5 impairs synthesis and utilization of starch in pollen grains and causes male sterility.

There is little known about the function of rice hexokinases (HXKs) in planta. We characterized hxk5-1, a Tos17 mutant of OsHXK5 that is up-regulated in maturing pollen, a stage when starch accumulates. Progeny analysis of self-pollinated heterozygotes of hxk5-1 and reciprocal crosses between the wild-type and heterozygotes revealed that loss of HXK5 causes male sterility. Homozygous hxk5-1, produced via anther culture, and additional homozygous hxk5-2, hxk5-3 and hxk5-4 lines created by CRISPR/Cas9 confirmed the male-sterile phenotype. In vitro pollen germination ability and in vivo pollen tube growth rate were significantly reduced in the hxk5 mutant pollen. Biochemical analysis of anthers with the mutant pollen revealed significantly reduced hexokinase activity and starch content, although they were sufficient to produce some viable seed. However, the mutant pollen was unable to compete successfully against wild-type pollen. Expression of the catalytically inactive OsHXK5-G113D did not rescue the hxk5 male-sterile phenotype, indicating that its catalytic function was responsible for pollen fertility, rather than its role in sugar sensing and signaling. Our results demonstrate that OsHXK5 contributes to a large portion of the hexokinase activity necessary for the starch utilization pathway during pollen germination and tube growth, as well as for starch biosynthesis during pollen maturation.

Profiling of the Major Phenolic Compounds and Their Biosynthesis Genes in Sophora flavescens Aiton.

Sophorae Radix (Sophora flavescens Aiton) has long been used in traditional medicine in East Asia due to the various biological activities of its secondary metabolites. Endogenous contents of phenolic compounds (phenolic acid, flavonol, and isoflavone) and the main bioactive compounds of Sophorae Radix were analyzed based on the qualitative HPLC analysis and evaluated in different organs and at different developmental stages. In total, 11 compounds were detected, and the composition of the roots and aerial parts (leaves, stems, and flowers) was significantly different. trans-Cinnamic acid and p-coumaric acid were observed only in the aerial parts. Large amounts of rutin and maackiain were detected in the roots. Four phenolic acid compounds (benzoic acid, caffeic acid, ferulic acid, and chlorogenic acid) and four flavonol compounds (kaempferol, catechin hydrate, epicatechin, and rutin) were higher in aerial parts than in roots. To identify putative genes involved in phenolic compounds biosynthesis, a total of 41 transcripts were investigated. Expression patterns of these selected genes, as well as the multiple isoforms for the genes, varied by organ and developmental stage, implying that they are involved in the biosynthesis of various phenolic compounds both spatially and temporally.

Role of rice cytosolic hexokinase OsHXK7 in sugar signaling and metabolism.

We characterized the function of the rice cytosolic hexokinase OsHXK7 (Oryza sativa Hexokinase7), which is highly upregulated when seeds germinate under O2 -deficient conditions. According to transient expression assays that used the promoter:luciferase fusion construct, OsHXK7 enhanced the glucose (Glc)-dependent repression of a rice α-amylase gene (RAmy3D) in the mesophyll protoplasts of maize, but its catalytically inactive mutant alleles did not. Consistently, the expression of OsHXK7, but not its catalytically inactive alleles, complemented the Arabidopsis glucose insensitive2-1 (gin2-1) mutant, thereby resulting in the wild type characteristics of Glc-dependent repression, seedling development, and plant growth. Interestingly, OsHXK7-mediated Glc-dependent repression was abolished in the O2 -deficient mesophyll protoplasts of maize. This result provides compelling evidence that OsHXK7 functions in sugar signaling via a glycolysis-dependent manner under normal conditions, but its signaling role is suppressed when O2 is deficient. The germination of two null OsHXK7 mutants, oshxk7-1 and oshxk7-2, was affected by O2 deficiency, but overexpression enhanced germination in rice. This result suggests the distinct role that OsHXK7 plays in sugar metabolism and efficient germination by enforcing glycolysis-mediated fermentation in O2 -deficient rice.

Development of a simple and efficient system for excising selectable markers in Arabidopsis using a minimal promoter::Cre fusion construct.

The development of rapid and efficient strategies to generate selectable marker-free transgenic plants could help increase the consumer acceptance of genetically modified (GM) plants. To produce marker-free transgenic plants without conditional treatment or the genetic crossing of offspring, we have developed a rapid and convenient DNA excision method mediated by the Cre/loxP recombination system under the control of a -46 minimal CaMV 35S promoter. The results of a transient expression assay showed that -46 minimal promoter::Cre recombinase (-46::Cre) can cause the loxP-specific excision of a selectable marker, thereby connecting the 35S promoter and ß-glucuronidase (GUS) reporter gene. Analysis of stable transgenic Arabidopsis plants indicated a positive correlation between loxP-specific DNA excision and GUS expression. PCR and DNA gel-blot analysis further revealed that nine of the 10 tested T(1) transgenic lines carried both excised and nonexcised constructs in their genomes. In the subsequent T(2) generation plants, over 30% of the individuals for each line were marker-free plants harboring the excised construct only. These results demonstrate that the -46::Cre fusion construct can be efficiently and easily utilized for producing marker-free transgenic plants.

Identification and characterization of the duplicate rice sucrose synthase genes OsSUS5 and OsSUS7 which are associated with the plasma membrane.

Systematic searches using the complete genome sequence of rice (Oryza sativa) identified OsSUS7, a new member of the rice sucrose synthase (OsSUS) gene family, which shows only nine single nucleotide substitutions in the OsSUS5 coding sequence. Comparative genomic analysis revealed that the synteny between OsSUS5 and OsSUS7 is conserved, and that significant numbers of transposable elements are scattered at both loci. In particular, a 17.6-kb genomic region containing transposable elements was identified in the 5' upstream sequence of the OsSUS7 gene. GFP fusion experiments indicated that OsSUS5 and OsSUS7 are largely associated with the plasma membrane and partly with the cytosol in maize mesophyll protoplasts. RT-PCR analysis and transient expression assays revealed that OsSUS5 and OsSUS7 exhibit similar expression patterns in rice tissues, with the highest expression evident in roots. These results suggest that two redundant genes, OsSUS5 and OsSUS7, evolved via duplication of a chromosome region and through the transposition of transposable elements.

Expression analysis and functional characterization of the monosaccharide transporters, OsTMTs, involving vacuolar sugar transport in rice (Oryza sativa).

In Arabidopsis, the compartmentation of sugars into vacuoles is known to be facilitated by sugar transporters. However, vacuolar sugar transporters have not been studied in detail in other plant species. To characterize the rice (Oryza sativa) tonoplast monosaccharide transporters, OsTMT1 and OsTMT2, we analysed their subcellular localization using green fluorescent protein (GFP) and expression patterns using reverse-transcription polymerase chain reaction (RT-PCR), performed histochemical beta-glucuronidase (GUS) assay and in situ hybridization analysis, and assessed sugar transport ability using isolated vacuoles. Expression of OsTMT-GFP fusion protein in rice and Arabidopsis revealed that the OsTMTs localize at the tonoplast. Analyses of OsTMT promoter-GUS transgenic rice indicated that OsTMT1 and OsTMT2 are highly expressed in bundle sheath cells, and in vascular parenchyma and companion cells in leaves, respectively. Both genes were found to be preferentially expressed in the vascular tissues of roots, the palea/lemma of spikelets, and in the main vascular tissues and nucellar projections on the dorsal side of the seed coats. Glucose uptake studies using vacuoles isolated from transgenic mutant Arabidopsis (tmt1-2-3) expressing OsTMT1 demonstrated that OsTMTs are capable of transporting glucose into vacuoles. Based on expression analysis and functional characterization, our present findings suggest that the OsTMTs play a role in vacuolar glucose storage in rice.

Role of the rice hexokinases OsHXK5 and OsHXK6 as glucose sensors.

The Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) is recognized as an important glucose (Glc) sensor. However, the function of hexokinases as Glc sensors has not been clearly demonstrated in other plant species, including rice (Oryza sativa). To investigate the functions of rice hexokinase isoforms, we characterized OsHXK5 and OsHXK6, which are evolutionarily related to AtHXK1. Transient expression analyses using GFP fusion constructs revealed that OsHXK5 and OsHXK6 are associated with mitochondria. Interestingly, the OsHXK5DeltamTP-GFP and OsHXK6DeltamTP-GFP fusion proteins, which lack N-terminal mitochondrial targeting peptides, were present mainly in the nucleus with a small amount of the proteins seen in the cytosol. In addition, the OsHXK5NLS-GFP and OsHXK6NLS-GFP fusion proteins harboring nuclear localization signals were targeted predominantly in the nucleus, suggesting that these OsHXKs retain a dual-targeting ability to mitochondria and nuclei. In transient expression assays using promoterluciferase fusion constructs, these two OsHXKs and their catalytically inactive alleles dramatically enhanced the Glc-dependent repression of the maize (Zea mays) Rubisco small subunit (RbcS) and rice alpha-amylase genes in mesophyll protoplasts of maize and rice. Notably, the expression of OsHXK5, OsHXK6, or their mutant alleles complemented the Arabidopsis glucose insensitive2-1 mutant, thereby resulting in wild-type characteristics in seedling development, Glc-dependent gene expression, and plant growth. Furthermore, transgenic rice plants overexpressing OsHXK5 or OsHXK6 exhibited hypersensitive plant growth retardation and enhanced repression of the photosynthetic gene RbcS in response to Glc treatment. These results provide evidence that rice OsHXK5 and OsHXK6 can function as Glc sensors.